WO-A1-85/05553 discloses bacterial cell surface proteins having fibronectin, fibrinogen, collagen, and/or laminin binding ability. Thereby it is shown that different bacteria have an ability to bind to fibronectin, fibrinogen, collagen, and/or laminin. It is further shown that fibronectin binding protein has a molecular weight of 165 kD and/or 87 kD, whereby it is probable that the smaller protein is a part of the larger one.
Fibronectin is a disulfide linked dimeric glycoprotein (M=450,000) which is present in a soluble form in blood plasma and other body fluids, and is deposited in a fibrillar form as a major constituent of the extracellular matrix of loose connective tissue. It is composed of three different structural motifs, termed type I, II, and III homologies (Petersen et al., 1983; PNAS), resulting in a modular organization cf the fibronectin molecule in which its several biological activities can each be attributed to specific domains.
The major biological role of fibronectin appears to be related to its ability to mediate the adhesion of eukaryotic cells to the extracellular matrix through a specific interaction between discrete cell surface receptors, and a 105-kDa central region of the molecule composed entirely of type III homology units (Pierschbacher and Ruoslahti, 1984, Nature 309:30-33). The cell binding domain is succeeded at its C-terminal end by a 31 kDa heparin binding domain and a 30 kDa fibrin binding domain. Preceding the cell binding domain is a 42 kDa gelatin (collagen) binding domain, and an N-terminal 29 kDa domain consisting of five consecutive type I repeats that binds fibrin, heparin and bacteria (Yamada 1983, Ann. Rev. Biochem. 52:761-799).
Several pathogenic Gram-positive staphylococci and streptococci have been reported to bind exclusively to the 29-kDa N-terminal domain of fibronectin (Speziale et al., 1984, J. Bacteriol. 157:420-427, Mosher and Proctor, 1980 Science 209:927-929). Some of the Gram-negative enteric bacteria, namely Escherichia coli, Salmonella typhimurium, S. enteritidis, and S. dublin have also been reported to bind fibronectin (Froman et al., 1984, JBC 259:14899-14905; Van de Water et al., 1983, Science 220:201-204; Faris et al., 1986, FEM Microbiol. Lett. 34:221-224; Baloda et al., 1986, FEMS Microbiol. Lett. 34:225-229; Batoda et al., 1985, FEMS Microbiol. Lett. 28:1-5; Kristiansen et al., 1987), and it was reported for E. coli, that the bacteria bound to the 29 kDa N-terminal domain, and a second unidentified site (Froman et al., 1984). In contrast, the spirochete Treponema palladium, and the trypanosome Trypanosoma cruzi differ from other fibronectin binding microorganisms in showing a specificity for the 105 kDa eukaryotic cell binding domain (Thomas et al., 1985a, J. Exp. Med. 161:514-525; Ouaissi et al., 1986, J. Exp. Med. 162:1715-1719).
The dissociation constants for the interaction of these microorganisms with fibronectin show a 10.sup.2 to 10.sup.3 -fold higher affinity than that reported for the binding of intact fibronectin to human fibroblasts (Hook et al , 1989; Akiyama and Yamada, 1985, JBC 260:4492.4500). This avidity for fibronectin may be a virulence factor, facilitating the colonization of wound tissues and blood clots, where a fibronectin rich matrix is known to be deposited during the first and second weeks of wound repair (Kurikinen et al., 1980, Lab Invest. 43:47-51; Grinell et al., 1981, J. Invest. Dermatol. 76:181-189; Clark et al., 1982, J. Invest. Dermatol. 79:269-276). It has been the intent of the present research to characterize microbial fibronectin binding proteins (FnBPs), and to develop receptor analogs for potential therapeutic application towards preventing or reducing the risk of wound infection.
Studies on FnBPs have so far mostly dealt with the FnBP of S. aureus. A FnBP with an M=of 210,000 has been reported from three laboratories (Espersen and Clemmensen, 1982, Infect. Immun. 37:526-531; Froman et al., 1987, J. Biol. Chem. 262:6564-6571; Signas et al., 1989, PNAS 86:699-703). The gene encoding the FnBP of S. aureus 8325-4 has been cloned and expressed in E. coli (Flock et al., 1987, EMBO J. 2351-2357). The fibronectin binding activity was localized to a 600 base pair insert, which when fused in frame with DNA coding for two IgG binding domains of staphylococcal protein A, yielded a protein fusion designated ZZ-FR which had the same fibronectin binding activity as the native 210 kDa receptor. Subsequent DNA sequence analysis revealed 600 base pair insert to code for 184 amino acids, of which a prominent feature was a 38 amino acid homology unit repeated three times, and partially a fourth (Signas et al., 1989).
It has been found that synthetic peptide analogs to each the homology units, designated D1, D2, and D3, were effective inhibitors of fibronectin binding to S. aureus 8325-4. Peptide D3 constructed on the third homology unit was 50 to 100-fold more effective-as an inhibitor of fibronectin binding, but showed a considerable deviation from the basic homology unit. In the present invention it is further defined the fibronectin binding determinant within the D3 homology unit through means of chemical modification, proteolytic cleavage, and chemical synthesis of a number smaller peptides encompassing the D3 sequence.